Project Summary Globally, the number of people with type 2 diabetes mellitus (T2D) is approaching 350 million [1]. With this number rapidly rising, the urgent need for effective therapeutics to treat this disease is also increasing. Research efforts have produced significant advances in treatment options, including the introduction of dipeptidyl peptidase 4 (DPP4) inhibitors, glucagon-like peptide-1 (GLP-1) mimetics and SGLT2 inhibitors to improve glucose-stimulated insulin secretion and lower blood glucose. Despite these advances, there remains a gap in the normalization of glucose and the prevention of complications among the population that current treatments have been unable to address, leaving millions of patients with insufficient and unsafe levels of glucose control. Recently, a protease enzyme called fibroblast activation protein (FAP) has emerged as a potential target for the treatment of T2D and obesity [2]. FAP is the most closely related enzyme to DPP4 [3, 4], a validated drug target. Multiple pharmacological inhibitors of DPP4 are now widely used in patients with T2D [5]. Previous interest in FAP has focused on its potential as a target for cancer therapy due to its upregulation in epithelial tumors [6]. However, recent FAP-specific activity assays have identified FAP activity in the circulation, as well as in several tissues with roles in metabolic regulation [7]. Most intriguingly, preliminary characterization of a global FAP knockout (Fap-/-) mouse demonstrated a) significant protection against diet-induced obesity and b) improved glycemia, phenotypic findings similar to those described in the global DPP4 knockout (Dpp4-/-) mouse [2]. Importantly, FAP has a unique endopeptidase activity, suggesting that its enzymatic targets are different from those of DPP4. Thus, FAP inhibition may provide a novel route to treatment of diabetes. We have independently generated mutant Fap-/- mice, enabling us to determine the metabolic phenotype ensuing from genetic FAP deficiency. Complementary experiments will utilize a potent and specific FAP inhibitor, compound 5057, to determine whether pharmacological FAP inhibition produces the same metabolic benefits observed in Fap-/- mice. Fap-/- mice and wild-type mice treated with compound 5057 will be employed to identify physiological mechanisms (and FAP substrates) underlying the metabolic benefits of FAP inactivation. We are hopeful that this research will lay the groundwork for clinical testing of a new class of drugs for the treatment of T2D.